Abstract:

A graft prostheses (11), materials and method for implanting,
transplanting, replacing, or repairing a part of a patient. The graft
prosthesis includes a purified, collagen-based matrix structure removed
from a submucosa tissue source. The submucosa tissue source is purified
by disinfection and removal steps to deactivate and remove contaminants,
thereby making the purified structure biocompatible and suitable for
grafting on and/or in a patient.

22. The graft prosthesis of claim 1, wherein said purified structure
comprises a disinfected and then delaminated submucosa tissue source.

23. A graft prosthesis comprising:a purified, collagen-based matrix
structure removed from a submucosa tissue source, said purified structure
having an endotoxin level of less than 12 endotoxin units per gram.

24. A graft prosthesis comprising:a purified, collagen-based matrix
structure removed from a submucosa tissue source, said purified structure
having a nucleic acid content level of less than 2 micrograms per
milligram.

25. A graft prosthesis comprising:a purified, collagen-based matrix
structure removed from a submucosa tissue source, said purified structure
having a virus level of less than 500 plaque forming units per gram.

26. A graft prosthesis comprising:a purified, collagen-based matrix
structure removed from a submucosa tissue source, said purified structure
having a processing agent level of less than 100,000 parts per million
per kilogram.

27. A method for obtaining a collagen-based matrix from a submucosa tissue
source, comprising:treating the submucosa tissue source with a
disinfecting agent to provide a disinfected submucosa tissue source;
andremoving the collagen-based matrix from the disinfected submucosa
tissue source.

28. The method of claim 27, wherein the submucosa tissue source is from an
alimentary tract of a mammal.

29. The method of claim 28, wherein the mammal is a pig.

30. The method of claim 29, wherein the submucosa tissue source is from
the small intestine of a pig.

31. The method of claim 27, wherein the disinfecting agent is an oxidizing
agent.

32. The method of claim 27, wherein the disinfecting agent is a peroxy
compound.

33. The method of claim 32, wherein the disinfecting agent is an organic
peroxy compound.

34. The method of claim 33, wherein the disinfecting agent is a peracid.

35. The method of claim 34, wherein the peracid is selected from the group
consisting of peracetic acid, perpropionic acid and perbenzoic acid.

36. The method of claim 35, wherein the peracid is peracetic acid.

37. The method of claim 34, wherein said treating includes treating the
submucosa tissue source with a medium containing an alcohol and the
peracid.

38. The method of claim 37, wherein the alcohol has one to about six
carbon atoms.

39. The method of claim 38, wherein the alcohol is selected from the group
consisting of ethanol, propanols, and butanols.

40. The method of claim 39, wherein the alcohol is ethanol.

41. The method of claim 40, wherein the medium is an aqueous ethanol
solution containing from about 0.1% to about 0.3% by volume peracetic
acid.

42. The method of claim 34, wherein said treating includes treating the
submucosa tissue source with a medium containing the peracid and having a
pH of about 2 to about 6.

43. The method of claim 42, wherein the medium has a pH of about 2 to
about 4.

44. The method of claim 43, wherein the peracid is peracetic acid, and the
medium contains about 0.1% to about 0.3% by volume of peracetic acid.

45. A method for obtaining a collagen-based matrix from a submucosa tissue
source, comprising:providing a submucosa tissue source which has been
treated with a disinfecting agent; andremoving the collagen-based matrix
from said submucosa tissue source.

46. The method of claim 45, wherein said submucosa tissue source is from a
small intestine.

47. The method of claim 46, wherein said disinfecting includes treating
the submucosa tissue source with an oxidizing agent.

48. The method of claim 47, wherein said treating includes contacting the
submucosa tissue source with an aqueous medium containing the oxidizing
agent.

49. The method of claim 47, wherein said treating includes contacting the
submucosa tissue source with an aqueous medium containing a peroxy
compound.

50. The method of claim 49, wherein the peroxy compound is a peracid.

51. The method of claim 50, wherein the peracid is peracetic acid.

52. The process of claim 51, wherein the medium comprises an alcohol.

53. The process of claim 52, wherein the alcohol is ethanol.

54. The method of claim 51, wherein the small intestine is from a pig.

55. A composition comprising:a collagen-containing structure removed from
a tissue source initially containing said structure and other tissue,
said collagen-containing structure having an endotoxin level of no
greater than 12 endotoxin units per gram.

56. The composition of claim 55, wherein said collagen-containing layer is
submucosa and said tissue source is small intestine.

57. The composition of claim 56, wherein said tissue source is pig small
intestine.

58. The composition of claim 55, wherein said endotoxin level is less than
10 endotoxin units per gram.

59. The composition of claim 58, wherein said endotoxin level is less than
5 endotoxin units per gram.

60. The composition of claim 50, wherein said endotoxin level is less than
1 endotoxin unit per gram.

61. The composition of claim 60, wherein said endotoxin level is less than
0.5 endotoxin units per gram.

62. A purified collagen-containing matrix obtained from a mammalian tissue
source, said matrix comprising mammalian tela submucosa and residual
contaminants from said mammalian tissue source, said structure obtainable
by a process which comprises disinfecting said mammalian tissue and then
removing said structure from the disinfected mammalian tissue.

63. The composition of claim 62 wherein said disinfecting includes
contacting the mammalian tissue source with an aqueous solution
containing a peracid.

64. The composition of claim 63 wherein the peracid is peracetic acid.

Description:

TECHNICAL FIELD

[0001]This invention relates generally to a medical structure and, in
particular, to a graft prosthesis, materials, and methods therefor.

BACKGROUND OF THE INVENTION

[0002]Tissue implants in a purified form and derived from collagen-based
materials have been manufactured and disclosed in the literature.
Cohesive films of high tensile strength have been manufactured using
collagen molecules or collagen-based materials. Aldehydes, however, have
been generally utilized to cross-link the collagen molecules to produce
films having high tensile strengths. With these types of materials, the
aldehydes may leech out of the film, e.g. upon hydrolysis. Because such
residues are cytotoxic, the films are poor tissue implants.

[0003]Other techniques have been developed to produce collagen-based
tissue implants while avoiding the problems associated with aldehyde
cross-linked collagen molecules. One such technique is illustrated in
U.S. Pat. No. 5,141,747 wherein the collagen molecules are cross-linked
or coupled at their lysine epsilon amino groups followed by denaturing
the coupled, and preferably modified, collagen molecules. The disclosed
use of such collagen material is for tympanic membrane repair. While such
membranes are disclosed to exhibit good physical properties and to be
sterilized by subsequent processing, they are not capable of remodeling
or generating cell growth or, in general, of promoting regrowth and
healing of damaged or diseased tissue structures.

[0004]In general, researchers in the surgical arts have been working for
many years to develop new techniques and materials for use as implants
and grafts to replace or repair damaged or diseased tissue structures,
for example, blood vessels, muscle, ligaments, tendons and the like. It
is not uncommon today, for instance, for an orthopedic surgeon to harvest
a patellar tendon of autogenous or allogenous origin for use as a
replacement for a torn cruciate ligament. The surgical methods for such
techniques are known. Further, it has been common for surgeons to use
implantable prostheses formed from plastic, metal and/or ceramic material
for reconstruction or replacement of physiological structures. Yet,
despite their wide use, surgical implanted prostheses present many
attendant risks to the patient.

[0005]Researchers have also been attempting to develop satisfactory
polymer or plastic materials to serve as such functional tissue
structures and/or other connective tissues, e.g., those Involved in
hernia and joint dislocation injuries. It has been discovered that it is
difficult to provide a tough, durable plastic material which is suitable
for long term connective tissue replacement. The tissues surrounding the
plastic material can become infected and difficulties in treating such
infections often lead to the failure of the implant or prostheses.

[0006]As mentioned above, various collagen-based materials have also been
utilized for the above-mentioned tissue replacements; however, these
materials either did not exhibit the requisite tensile strength or also
had problems with infection and other immunogenic responses,
encapsulation, or had other problems when they may have been loaded with
antibiotics, growth factors and the like. For example, U.S. Pat. No.
4,956,178 discloses a submucosa collagen matrix which is obtained from
the intestinal tract of mammals; however, it is disclosed that the
collagen matrix is loaded with antibiotics. In a related patent, U.S.
Pat. No. 5,372,821, it is disclosed that a submucosa collagen matrix may
be sterilized by conventional techniques, e.g., aldehyde tanning,
propylene oxide, gamma radiation and peracetic acid. No specific
processing steps are disclosed except that the submucosa layer is first
delaminated from the surrounding tissue prior to sterilization treatment.

[0007]Therefore, there is a need to obtain improved purified forms of
collagen-based matrices from tissue sources thereof. Also, there is a
need to provide a process whereby the ease of removal of such matrices
from tissue sources is enhanced so as to yield such improved, purified
products. The present invention is addressed to these needs.

SUMMARY OF THE INVENTION

[0008]In accordance with one preferred embodiment of the present
invention, provided is a graft prosthesis which includes a purified,
collagen-based matrix structure removed from a submucosa tissue source,
wherein the purified structure has a contaminant level making the
purified structure biocompatible.

[0009]Another preferred embodiment of the invention provides a graft
prosthesis which includes a purified, collagen-based matrix structure
removed from a submucosa tissue source, wherein the purified structure
has an endotoxin level of less than 12 endotoxin units per gram.

[0010]Another preferred embodiment of the invention provides a graft
prosthesis which includes a purified, collagen-based matrix structure
removed from a submucosa tissue source, wherein the purified structure
has a nucleic acid content level of less than 2 micrograms per milligram.

[0011]Another preferred embodiment of the invention provides a graft
prosthesis including a purified, collagen-based matrix structure removed
from a submucosa tissue source, wherein the purified structure has a
virus level of less than 500 plaque forming units per gram.

[0012]The present invention also provides a graft prosthesis which
includes a purified, collagen-based matrix structure removed from a
submucosa tissue source, wherein the purified structure has a processing
agent level of less than 100,000 parts per million per kilogram.

[0013]A further embodiment of the invention concerns a method for
obtaining a collagen-based matrix from a submucosa tissue source. The
method includes treating the submucosa tissue source with a disinfecting
agent to provide a disinfected submucosa tissue source, and removing the
collagen-based matrix from the disinfected submucosa tissue source.

[0014]Another preferred embodiment of the invention provides a method for
obtaining a collagen-based matrix from a submucosa tissue source, which
includes providing a submucosa tissue source which has been treated with
a disinfecting agent, and removing the collagen-based matrix from the
submucosa tissue source.

[0015]The present invention also concerns a composition which includes a
collagen-containing structure removed from a tissue source initially
containing the structure and other tissue, wherein the
collagen-containing structure has an endotoxin level of no greater than
12 endotoxin units per gram.

[0016]Also provided by the present invention is a purified
collagen-containing matrix obtained from a mammalian tissue source, the
matrix including mammalian tela submucosa and being obtainable by a
process which includes disinfecting the mammalian tissue source then
removing the structure from the resulting disinfected mammalian tissue
source.

[0017]In preferred aspects, the invention provides purified forms of tela
submucosa collagen matrices derived from the alimentary, respiratory,
urinary or genital tracts of animals, the matrices having a bioburden
level of substantially zero, and/or being essentially free of pyrogens. A
preferred collagen matrix is capable of being implanted within a human or
animal patient without causing a cytotoxic response, infection, rejection
of the implant or any other harmful effect in a majority of patients.
While a preferred implantable collagen matrix according to some aspects
of the present invention comprises primarily tela submucosa, the collagen
matrix in this instance may also comprise partial layers of laminar
muscularis mucosa, muscularis mucosa, lamina propria, a stratum compactum
layer and/or other such tissue materials depending upon the source from
which it was derived.

[0018]Further in accordance with the present invention, a purified
delaminated tela submucosa collagen matrix is provided which is derived
from the alimentary, respiratory, urinary or genital tracts of animals or
humans, wherein said purified submucosa collagen matrix is produced by
delaminating a disinfected tela submucosa source to obtain the
delaminated tela submucosa collagen matrix. An advantageous matrix may be
obtained, for example, by a process comprising treating an unprocessed,
undelaminated tela submucosa source harvested from the alimentary,
respiratory, urinary or genital tracts of animals with a disinfecting
agent, followed by delaminating the tela submucosa collagen matrix from
the attached tissues. The preferred collagen matrix has a bioburden level
of substantially zero and capable of being implanted within a human or
animal patient without causing a cytotoxic response, infection, rejection
of the implant or any other harmful effect in a majority of patients.

[0019]Still further in accordance with the present invention, a method is
provided for obtaining a highly pure, delaminated tela submucosa collagen
matrix in a substantially sterile state, comprising delaminating a
disinfected tela submucosa tissue source to obtain the delaminated tela
submucosa collagen matrix. A preferred method comprises treating an
undelaminated tela submucosa source harvested from the alimentary,
respiratory, urinary or genital tracts of animals or humans with a
disinfecting agent, followed by delaminating the tela submucosa from its
other source tissues attached to the tela submucosa.

[0020]Still further in accordance with the present invention, provided is
a highly pure tela submucosa collagen matrix derived from the alimentary,
respiratory, urinary or genital tracts of animals having a bioburden of
substantially zero, and wherein said tela submucosa collagen matrix
contains substantially no surface debris, e.g. including substantially no
muscle tissue, mucosal layers, lipids or cellular debris. The preferred
collagen matrix is capable of being implanted within a human or animal
patient without causing cytotoxic response, infection, rejection of the
implant or any other harmful effect to the patient.

[0021]Still further in accordance with the present invention, a highly
pure tela submucosa is provided which is derived from the alimentary,
respiratory, urinary or genital tracts of animals and wherein the tela
submucosa is delaminated in a substantially sterile condition comprising
growth factors, and is produced by rinsing the delaminated, tela
submucosa source with a solvent, for instance water, followed by
treatment with a disinfecting agent, preferably a peracid, at a pH of
about 1.5 to about 10 followed by delamination of the tela submucosa from
the attached tissues. The peracid is buffered at pH levels greater than
7. Desirably, collagen matrices so produced have a substantial high
content of one or more growth factors.

[0022]Still further in accordance with the present invention, provided is
a tissue graft composition which includes a tela submucosa collagen
matrix which is essentially pyrogen free. More preferred such
compositions will include a tela submucosa collagen matrix which has a
pyrogen content of about 1 endotoxin unit per gram (EU/g) or less.

[0023]Still further in accordance with the present invention, a highly
pure, tela submucosa as described above, will demonstrate active
angiogenesis in vivo upon implantation in a human or animal patient.

[0024]This invention relates to purified, implantable tissue constructs, a
process for producing such purified, implantable tissue constructs, and
their use to promote regrowth and healing of damaged or diseased tissue
structures. More particularly, the invention is directed to purified
forms of tela submucosa collagen matrix suitable for use as an
implantable tissue, and methods for producing such purified forms of this
collagen-based implantable tissue.

[0025]These and other aspects of the present invention will become
apparent to those skilled in the art upon reviewing the specification
that follows.

BRIEF DESCRIPTION OF THE DRAWING

[0026]FIG. 1 provides a perspective view of a tubular graft prosthesis
structure in accordance with the invention.

DETAILED DESCRIPTION

[0027]For the purpose of promoting an understanding of the principles of
the invention, reference will now be made to certain preferred
embodiments thereof and specific language will be used to describe the
same. It will nevertheless be understood that no limitation of the scope
of the invention is thereby intended, such alterations, further
modifications and applications of the principles of the invention as
described herein being contemplated as would normally occur to one
skilled in the art to which the invention relates.

[0028]In the discussions herein, a number of terms are used. In order to
provide and clear and consistent understanding of the specification and
claims, the following definitions are provided.

[0029]Bioburden--refers to the number of living microorganisms, reported
in colony-forming units (CFU), found on and/or in a given amount of
material. Illustrative microorganisms include bacteria, fungi and their
spores.

[0030]Disinfection--refers to a reduction in the bioburden of a material.

[0031]Sterile--refers to a condition wherein a material has a bioburden
such that the probability of having one living microorganism (CFU) on
and/or in a given section of the material is one in one-million or less.

[0032]Pyrogen--refers to a substance which produces febrile response after
introduction into a host.

[0033]Endotoxin--refers to a particular pyrogen which is part of the cell
wall of gram-negative bacteria. Endotoxins are continually shed from the
bacteria and contaminate materials.

[0034]Purification--refers to the treatment of a material to remove one or
more contaminants which occur with the material, for instance
contaminants with which the material occurs in nature, and/or
microorganisms or components thereof occurring on the material.
Illustratively, the contaminants may be those known to cause toxicity,
infectivity, pyrogenicity, irritation potential, reactivity, hemolytic
activity, carcinogenicity and/or immunogenicity.

[0035]Biocompatibility--refers to the ability of a material to pass the
biocompatibility tests set forth in International Standards Organization
(ISO) Standard No. 10993 and/or the U.S. Pharmacopeia (USP) 23 and/or the
U.S. Food and Drug Administration (FDA) blue book memorandum No. G95-1,
entitled "Use of International Standard ISO-10993, Biological Evaluation
of Medical Devices Part-1: Evaluation and Testing." Typically, these
tests assay as to a material's toxicity, infectivity, pyrogenicity,
irritation potential, reactivity, hemolytic activity, carcinogenicity
and/or immunogenicity. A biocompatible structure or material when
introduced into a majority of patients will not cause an adverse reaction
or response. In addition, it is contemplated that biocompatibility can be
effected by other contaminants such as prions, surfactants,
oligonucleotides, and other biocompatibility effecting agents or
contaminants.

[0036]Contaminant--refers to an unwanted substance on, attached to, or
within a material. This includes, but is not limited to: bioburden,
endotoxins, processing agents such as antimicrobial agents, blood, blood
components, viruses, DNA, RNA, spores, fragments of unwanted tissue
layers, cellular debris, and mucosa.

[0037]Tela submucosa--refers to a layer of collagen-containing connective
tissue occurring under the mucosa in most parts of the alimentary,
respiratory, urinary and genital tracts of animals.

[0038]As disclosed above, the present invention generally provides graft
prostheses and materials including a purified collagen-based matrix
structure, and methods for obtaining and using the same. Advantageous
graft prostheses of the invention are obtained from a submucosa tissue
source, for example including animal tissues such as human or other
mammalian tissues, e.g. porcine, bovine or ovine tissues.

[0039]Tela submucosa, as with many animal tissues, is generally aseptic in
its natural state, provided the human or animal does not have an
infection or disease. This is particularly the case since the tela
submucosa is an internal layer within the alimentary, respiratory,
urinary and genital tracts of animals. Accordingly, it is generally not
exposed to bacteria and other cellular debris such as the epithelium of
the intestinal tract. One feature of the present invention is the
discovery that by disinfecting the source tissue for the tela submucosa
prior to delamination, the aseptic state of the tela submucosa layer can
be preserved or substantially preserved, particularly if the delamination
process occurs under sterile conditions.

[0040]In particular, it has been discovered that disinfecting the tela
submucosa source, followed by removal of a purified matrix including the
tela submucosa, e.g. by delaminating the tela submucosa from the tunica
muscularis and the tunica mucosa, minimizes the exposure of the tela
submucosa to bacteria and other contaminants. In turn, this enables
minimizing exposure of the isolated tela submucosa matrix to
disinfectants or sterilants if desired, thus substantially preserving the
inherent biochemistry of the tela submucosa and many of the tela
submucosa's beneficial effects.

[0041]A tela submucosa implantable collagen matrix according to the
present invention can, as indicated above, be obtained from the
alimentary, respiratory, urinary or genital tracts of animals.
Preferably, the tela submucosa tissues, which are collagen-based and thus
predominantly collagen, are derived from the alimentary tract of mammals
and most preferably from the intestinal tract of pigs. A most preferred
source of whole small intestine is harvested from mature adult pigs
weighing greater than about 450 pounds. Intestines harvested from
healthy, nondiseased animals Will contain blood vessels and blood supply
within the intestinal tract, as well as various microbes such as E. coli
contained within the lumen of the intestines. Therefore, disinfecting the
whole intestine prior to delamination of the tela submucosa substantially
removes these contaminants and provides a preferred implantable tela
submucosa tissue which is substantially free of blood and blood
components as well as any other microbial organisms, pyrogens or other
pathogens that may be present. In effect, this procedure is believed to
substantially preserve the inherent aseptic state of the tela submucosa,
although it should be understood that it is not intended that the present
invention be limited by any theory.

[0042]It is also desirable that the collagen matrix according to the
present invention be substantially free of any antibiotics, antiviral
agents or any antimicrobial type agents which may affect the inherent
biochemistry of the matrix and its efficacy upon implantation. In the
past, one method of treating such tissue material is to rinse the
delaminated tissue in saline and soak it in an antimicrobial agent, for
example, as disclosed in U.S. Pat. No. 4,956,178. While such techniques
can optionally be practiced with isolated submucosa of the present
invention, preferred processes according to present invention avoid the
use of antimicrobial agents and the like which may not only affect the
biochemistry of the collagen matrix but also can be unnecessarily
introduced into the tissues of the patient.

[0043]As discussed above, it has been discovered that a highly pure form
of an implantable tela submucosa collagen matrix may be obtained by first
disinfecting a tela submucosa source prior to removing a purified
collagen matrix including the tela submucosa layer, e.g. by delaminating
the tela submucosa source. It has also been discovered that certain
processing advantages as well as improved properties of the resultant
tela submucosa layer are obtained by this process, including greater ease
in removing attached tissues from the submucosa layer, and a
characteristic, low contaminant profile.

[0044]Processes of the invention desirably involve first rinsing the tela
submucosa source one or more times with a solvent, suitably water. The
rinsing step is followed by treatment with a disinfecting agent. The
disinfecting agent is desirably an oxidizing agent. Preferred
disinfecting agents are peroxy compounds, preferably organic peroxy
compounds, and more preferably peracids. Such disinfecting agents are
desirably used in a liquid medium, preferably a solution, having a pH of
about 1.5 to about 10, more preferably a pH of about 2 to about 6, and
most preferably a pH of about 2 to about 4. In methods of the present
invention, the disinfecting agent will generally be used under conditions
and for a period of time which provide the recovery of characteristic,
purified submucosa matrices as described herein, preferably exhibiting a
bioburden of essentially zero and/or essential freedom from pyrogens. In
this regard, desirable processes of the invention involve immersing the
tissue source (e.g. by submersing or showering) in a liquid medium
containing the disinfecting agent for a period of at least about 5
minutes, typically in the range of about 5 minutes to about 40 hours, and
more typically in the range of about 0.5 hours to about 5 hours.

[0045]A preferred peroxy disinfecting agent is hydrogen peroxide. The
concentration of hydrogen peroxide can range from about 0.05% to 30% by
volume. More preferably the hydrogen peroxide concentration is from about
1% to 10% by volume and most preferably from about 2% to 5% by volume.
The solution may or may not be buffered to a pH from about 5 to 9. More
preferably the pH is from about 6 to 7.5. These concentrations can be
diluted in water or in an aqueous solution of about 2% to about 30% by
volume alcohol. Most preferably the alcohol is ethanol. The solution
temperature can range from about 15 to 50° C. More preferably the
solution temperature is from about 20 to 40° C. Most preferably,
the solution temperature is from about 32 to 37° C. The exposure
time can range from about 10 to 400 minutes. Preferably, the exposure
time is from about 120 to 240 minutes. More preferably, the exposure time
is from 180 to 210 minutes.

[0046]A preferred organic peroxide disinfecting agent is perpropionic
acid. The concentration of perpropionic acid may range from about 0.1% to
10% by volume. More preferably the perpropionic acid concentration is
from about 0.1% to 1.0% by volume and most preferably from about 0.2% to
0.5% by volume. These concentrations of perpropionic acid can be diluted
in water or in an aqueous solution of about 2% to about 30% by volume
alcohol. Most preferably the alcohol is ethanol. The tela submucosa
tissue source can be exposed to the organic peroxide solution for periods
from about 15 minutes to about 40 hours, and more typically in the range
of about 0.5 hours to about 8 hours. Other peroxy disinfecting agents are
suitable for use as described in "Peroxygen Compounds", S. Block, in
Disinfection, Sterilization and Preservation, S. Block, Editor, 4th
Edition, Philadelphia, Lea & Febiger, pp. 167-181, 1991; and
"Disinfection with peroxygens", M. G. C. Baldry and J. A. L. Fraser, in
Industrial Biocides, K. Payne, Editor, New York, John Wiley and Sons, pp.
91-116, 1988.

[0047]Another oxidizing disinfecting agent is chlorhexidine
(1,6-di(4-chlorophenyldiguanido)hexane) in its digluconate form. The
concentration of chlorhexidine digluconate may range from about 0.1% to
15% by weight. More preferably, the chlorhexidine digluconate
concentration is from about 0.1% to 2% by weight and most preferably from
about 0.2% to 5% by weight. The solution may or may not be buffered to a
pH from about 5 to 8. More preferably the pH is from about 5.5 to 7.
These concentrations may be diluted in water or in an aqueous solution of
about 2% to about 20% by volume alcohol. Most preferably the alcohol is
ethanol at a concentration of about 5% to 10%. The solution temperature
may range from about 15 to 30° C. The exposure time may range from
about 10 to 400 minutes. More preferably the exposure time is from about
30 to 60 minutes. Other chlorine agents are described in "Chlorhexidine",
G. W. Denton, in Disinfection, Sterilization and Preservation, S. Block,
Editor, 4th Edition, Philadelphia, Lea & Febiger, pp. 274-289, 1991.

[0048]In preferred preparative processes, a peracid or other disinfecting
agent may be dissolved in a dilute aqueous alcohol solution, preferably
wherein the alcohol has from 1 to about 6 carbon atoms, and wherein the
alcohol may generally comprise from about 1% to about 30% by volume of
the solution. More preferred alcohols for use in the invention are
selected from the group consisting of ethanol, propanols and butanols.
Ethanol is a most preferred alcohol for these purposes.

[0049]When a peracid is used in the disinfection, it is preferably
selected from the group consisting of peracetic acid, perpropionic acid
or perbenzoic acid. Peracetic acid is the most preferred disinfecting
agent. The peracetic acid is preferably diluted into about a 2% to about
10% by volume alcohol solution. The concentration of the peracetic acid
may range, for example, from about 0.05% by volume to about 1.0% by
volume. Most preferably the concentration of the peracetic acid is from
about 0.1% to about 0.3% by volume. Hydrogen peroxide can also be used as
a disinfecting agent. Alternatively, or in addition, the tela submucosa
tissue source, e.g. from small intestine, may be disinfected utilizing
disinfecting agents such as glutaraldehyde, formalin and the like, which
are also known for their ability to introduce substantial crosslinking
into collagen matrices, in contrast to the action of other disinfecting
agents such as peracids which can be used to disinfect without
introducing such crosslinking. Additionally, the tela submucosa source
can be treated with radiation, e.g., gamma radiation, for purposes of
disinfection.

[0050]Variations on the disinfection process can also include the
following: [0051]1. Intestine is treated with 0.2% peracetic acid, 5%
ethanol solution at a ratio of 10:1 solution to intestine ratio by
weight. Solution has a pH of 2.6. Solution and intestine are vigorously
mixed for two hours. [0052]2. Intestine is treated with 1% peracetic
acid, 25% ethanol solution at a ration of 5:1 solution to intestine ratio
by weight. Solution has a pH of 2. Solution and intestine are vigorously
mixed for one hour. [0053]3. Intestine is treated with 1% peracetic acid,
15% ethanol, and 10% hydrogen peroxide solution at a ratio of 5:1
solution to intestine ratio by weight. Solution and intestine are
vigorously mixed for one hour. [0054]4. Whole small intestine is rinsed
four times with high purity water for 15 minutes. The intestine is then
subjected to 1.5 MRAD Electron Beam radiation. [0055]5. Whole small
intestine is rinsed four times with high purity water for 15 minutes.
Lengthwise along a conveyor belt, the intestine is subjected to
high-intensity pulsed light which disinfects the intestine.

[0056]Following the treatment as described above, the tela submucosa layer
is delaminated from its source, e.g., whole intestine, cow uterus and the
like. It has been found that by following this
post-disinfection-stripping procedure, it is easier to separate the tela
submucosa layer from the attached tissues, e.g. at least from attached
tunica muscularis tissue, as compared to stripping the tela submucosa
layer prior to disinfection. Moreover it has been discovered that the
resultant tela submucosa layer in its most preferred form exhibits
superior histology, in that there is less attached tissue and debris on
the surface compared to a tela submucosa layer obtained by first
delaminating the tela submucosa layer from its source and then
disinfecting the layer. Moreover, a more uniform tela submucosa tissue
can be obtained from this process, and a tela submucosa having the same
or similar physical and biochemical properties can be obtained more
consistently from each separate processing run. Importantly, a highly
purified, substantially sterile tela submucosa is obtained by this
process.

[0057]The stripping of the tela submucosa source is preferably carried out
by utilizing a disinfected or sterile casing machine, to produce a tela
submucosa which is substantially sterile and which has been minimally
processed. A suitable casing machine is the Model 3-U-400 Stridhs
Universal Machine for Hog Casing, commercially available from the AB
Stridhs Maskiner, Gotoborg, Sweden. Therefore, the measured bioburden
levels are minimal or substantially zero. Of course, other means for
delaminating the tela submucosa source can be employed without departing
from the present invention, including for example delaminating by hand.

[0058]It has also been discovered that more preferred processes according
to the present invention, not only will eliminate or significantly reduce
contaminants contained in the tela submucosa collagen matrix, but also
will produce a tissue which exhibits no substantial degradation of
physical and mechanical properties, e.g., differential porosity (i.e.
wherein one side of the submucosa layer has greater porosity than the
other side), and good strength, for example burst strength. Also, it has
been discovered that more preferred processes do not affect the
differential porosity of the tela submucosa collagen matrix which
ultimately affects the level of efficacy of this tissue implant. For
example, the tissue is not necessarily treated with a crosslinking agent
or a material that disrupts the porosity or inherent, native structure of
the collagen matrix. Moreover, when hydrogen peroxide is employed, the
matrix as a whole has greater porosity as well as a higher oxygen
content. This helps to ensure the absence of contaminants e.g.,
endotoxins, pyrogens and the like.

[0059]Also, in an advantageous form, the collagen-based matrices of the
present invention (e.g., including tela submucosa) demonstrate the
ability to induce active angiogenesis, i.e., an ingrowth of blood vessels
within the matrix of the tissue. In this regard, these preferred matrices
of the invention will contain beneficial components with which the
matrices naturally occur, including for example one or more of
glycosaminoglycans, glycoproteins, proteoglycans, and/or growth factors
(e.g. Transforming Growth Factor-Transforming Growth Factor-, and/or
Fibroblast Growth Factor 2 (basic)).

[0060]Preferred collagen-based matrices of the invention, preferably
submucosa-containing matrices, are also characterized by the low
contaminant levels set forth in Table 1 below, each contaminant level
taken individually or in any combination with some or all of the other
disclosed contaminant levels. The abbreviations in Table 1 are as
follows: CFU/g=colony forming units per gram; PFU/g=plaque forming units
per gram; ig/mg=micrograms per milligram; ppm/kg=parts per million per
kilogram.

[0061]Even more preferred collagen-based matrices of the invention contain
an endotoxin level of less than 1 EU/g, and most preferably less than 0.5
EU/g.

[0062]Purified collagen-based matrices according to the present invention
may be processed in a number of ways, to provide collagenous matrices
useful both in vitro and in vivo. For example, the submucosa may be
configured to provide tissue grafts useful in vascular applications, e.g.
as generally described in U.S. Pat. Nos. 2,127,903 and 4,902,508. With
reference now to FIG. 1, for use in vascular grafting, a generally
tubular graft prosthesis structure 11 is formed with or including the
collagen-based matrix 12, the diameter "D" of which approximates that of
a recipient blood vessel. In one mode, this may be accomplished by
manipulating a tubular segment or sheet of the tela submucosa to define a
cylinder having a diameter "D" approximately the same as that of the
recipient blood vessel, and suturing, bonding or otherwise securing the
longitudinal seam 13 to form an appropriately-dimensioned tubular
vascular graft having a lumen 14 for passage of blood. In illustrative
preparative procedures, the graft is formed over a sterile rod or mandrel
having an outer diameter approximately equal to that of the vessel to be
grafted. For instance, the rod is introduced into the lumen of a tela
submucosa segment retaining its native, tubular form. Redundant tissue is
then gathered, and the desired lumen diameter achieved by suturing along
the length of the graft (for example, using two continuous suture lines
or a simple interrupted suture line), or by using other art-recognized
tissue securing techniques. Alternatively, a sheet of the inventive tela
submucosa is wrapped about the rod to form an overlapping seam, which can
be sutured, glued or otherwise secured, to provide the tubular graft
construct. In preferred forms, the inner, luminal surface of the graft
can be formed by the mucosal side of the tela submucosa.

[0063]The tela submucosa of the invention possesses mechanical properties
highly desirable for tissue graft materials in vascular applications,
including low porosity index, high compliance, and a high burst strength.
One skilled in the art will appreciate that the preferred tissue graft
material will be of low enough porosity to prevent intraoperative
hemorrhage and yet of high enough porosity to allow extension of a
newly-developed vasa vasorum through the graft material to nourish the
neointima and luminal surface.

[0064]Tela submucosa tissue of the present invention can also be processed
to provide fluidized compositions, for instance using techniques as
described in U.S. Pat. No. 5,275,826. In this regard, solutions or
suspensions of the tela submucosa can be prepared by comminuting and/or
digesting the tela submucosa with a protease (e.g. trypsin or pepsin),
for a period of time sufficient to solubilize the tissue and form
substantially homogeneous solution. The submucosa starting material is
desirably comminuted by tearing, cutting, grinding, shearing or the like.
Grinding the submucosa in a frozen or freeze-dried state is advantageous,
although good results can be obtained as well by subjecting a suspension
of pieces of the submucosa to treatment in a high speed blender and
dewatering, if necessary, by centrifuging and decanting excess waste. The
comminuted tela submucosa can be dried, for example freeze dried, to form
a powder. Thereafter, if desired, the powder can be hydrated, that is,
combined with water or buffered saline and optionally other
pharmaceutically acceptable excipients, to form a fluid tissue graft
composition, e.g. having a viscosity of about 2 to about 300,000 cps at
25EC. The higher viscosity graft compositions can have a gel or paste
consistency.

[0065]Fluidized tela submucosa of this invention finds use as an
injectable heterograft for tissues, for example, bone or soft tissues, in
need of repair or augmentation most typically to correct trauma or
disease-induced tissue defects. The present fluidized submucosa
compositions are also used advantageously as a filler for implant
constructs comprising, for example, one or more sheets of tela submucosa
formed into sealed (sutured) pouches for use in cosmetic or
trauma-treating surgical procedures.

[0066]In one illustrative preparation, tela submucosa prepared as
described herein is reduced to small pieces (e.g. by cutting) which are
charged to a flat bottom stainless steel container. Liquid nitrogen is
introduced into the container to freeze the specimens, which are then
comminuted while in the frozen state to form a coarse tela submucosa
powder. Such processing can be carried out, for example, with a manual
arbor press with a cylindrical brass ingot placed on top of the frozen
specimens. The ingot serves as an interface between the specimens and the
arbor of the press. Liquid nitrogen can be added periodically to the tela
submucosa specimens to keep them frozen.

[0067]Other methods for comminuting tela submucosa specimens can be
utilized to produce a tela submucosa powder usable in accordance with the
present invention. For example, tela submucosa specimens can be
freeze-dried and then ground using a manual arbor press or other grinding
means. Alternatively, tela submucosa can be processed in a high shear
blender to produce, upon dewatering and drying, a tela submucosa powder.

[0068]Further grinding of the tela submucosa powder using a prechilled
mortar and pestle can be used to produce consistent, more finely divided
product. Again, liquid nitrogen is used as needed to maintain solid
frozen particles during final grinding. The powder can be easily hydrated
using, for example, buffered saline to produce a fluidized tissue graft
material of this invention at the desired viscosity.

[0069]To prepare another preferred fluidized material, a tela submucosa
powder can be sifted through a wire mesh, collected, and subjected to
proteolytic digestion to form a substantially homogeneous solution. For
example, the powder can be digested with 1 mg/ml of pepsin (Sigma
Chemical Co., St. Louis Mo.) and 0.1 M acetic acid, adjusted to pH 2.5
with HCl, over a 48 hour period at room temperature. After this
treatment, the reaction medium can be neutralized with sodium hydroxide
to inactivate the peptic activity. The solubilized submucosa can then be
concentrated by salt precipitation of the solution and separated for
further purification and/or freeze drying to form a protease-solubilized
intestinal submucosa in powder form.

[0070]Fluidized tela submucosa compositions of this invention find wide
application in tissue replacement, augmentation, and/or repair. The
fluidized submucosal compositions can be used to induce regrowth of
natural connective tissue or bone in an area of an existent defect. By
injecting an effective amount of a fluidized submucosa composition into
the locale of a tissue defect or a wound in need of healing, one can
readily take advantage of the biotropic properties of the tela submucosa.

[0071]It is also possible to form large surface area constructs by
combining two or more tela submucosa segments of the invention, for
instance using techniques as described in U.S. Pat. No. 2,127,903 and/or
International Publication No. WO 96/32146, dated 17 Oct. 1996, publishing
International Application No. PCT/US96/04271, filed 5 Apr. 1996. Thus, a
plurality of tela submucosa strips can be fused to one another, for
example by compressing overlapping areas of the strips under dehydrating
conditions, to form an overall planar construct having a surface area
greater than that of any one planar surface of the individual strips used
to form the construct.

[0072]Tela submucosa of the invention can also be employed to prepare
tissue graft constructs useful in orthopedic soft tissue applications,
for example in tendon or ligament repair, employing techniques in the art
which have been applied to other naturally-derived or synthetic graft
materials. For instance, repair techniques as generally described in U.S.
Pat. Nos. 2,127,903 and 5,281,422 can be undertaken using tela submucosa
of the present invention.

[0073]For tendon and ligament replacement applications, a segment of the
tela submucosa can be preconditioned by longitudinal stretching to an
elongated length. For example, a tela submucosa segment can be
conditioned by the prolonged application of a load on the longitudinal
axis of the segment (e.g. by suspending a weight from the segment) for a
period of time sufficient to allow about 10% to about 20% elongation of
the tissue segment. The graft material can also be preconditioned by
stretching in the lateral dimension. The tela submucosa segment can then
be configured, alone or in combination with other segments, to a variety
of shapes to serve as a ligament or tendon replacement, or to substitute
for or patch a broken or severed tendon or ligament.

[0074]For such connective tissue grafting applications, the segment is
desirably configured to have a layered or multilayered configuration,
with at least the opposite end portions and/or opposite lateral portions
being formed to have multiple layers of the graft material to provide
reinforcement for attachment to physiological structures such as bone,
tendon, ligament, cartilage and muscle. In a ligament replacement
application, opposite ends will be attached to first and second bones,
respectively, the bones typically being articulated as in the case of a
knee joint. In a tendon replacement application, a first end of the graft
construct will be attached to a bone, and a second end will be attached
to a muscle.

[0075]As indicated above, in connective tissue applications, it will be
advantageous to form, manipulate or shape the end portions of the graft
construct to be attached, for example, to a bone structure, in a manner
that will reduce the possibility of graft tearing at the point of
attachment. For these purposes, the tela submucosa graft material can be
folded or partially everted to provide multiple layers for g ripping, for
example, with spiked washers or staples. Alternatively, a tela submucosa
segment can be folded back on itself to join the end portions to provide
a first connective portion to be attached, for example, to a first bone
and a bend in the intermediate portion to provide a second connective
portion to be attached to a second bone articulated with respect to the
first bone.

[0076]For example, one of the end portions of the tela submucosa graft can
be adapted to be pulled through a tunnel in, for example, the femur and
attached thereto, while the other of the end portions may be adapted to
be pulled through a tunnel in the tibia and attached thereto to provide a
substitute for the natural cruciate ligament, the segment being adapted
to be placed under tension between the tunnels to provide a ligament
function, i.e., a tensioning and position function provided by a normal
ligament.

[0077]Because grafts used in orthopedic applications are typically placed
under tension in their surgical installation, it is preferable to combine
two or even more tissue segments to provide a multi-ply (multi-layered)
graft construct. It is another object of the present invention,
therefore, to provide such grafts in which two or more submucosa segments
are arranged to have their end portions joined together with the joined
end portions and/or lateral portions adapted to be attached to a bone,
tendon, ligament or other physiological structure. One method for
providing a double segment can be to pull one tubular segment internally
within another segment to provide a double-walled tube, the joined ends
of which can be attached, for example, to a bone, tendon or ligament.
These doubled segments will provide enhanced tensile strength and retela
submucosatance to stretching under tension. In other forms, multiple tela
submucosa segments or strips can be arranged in a braided configuration,
for example a diamond or sashcord braided configuration, or in a mesh
configuration including multiple loops intercoupled to neighboring loops,
which usefully serve in ligament or tendon repair.

[0078]Tela submucosa of the present invention can also be used to provide
an orthopedic graft for use as connective tissue to hold fractured bone
pieces together and in proper orientation in the body, the tissue segment
being formed to serve as a fracture wrap about segments of fractured bone
and to be attached to the bone.

[0079]In still further orthopedic applications, tela submucosa of the
invention can be used to repair bone tissue, for instance using the
general techniques described in U.S. Pat. No. 5,641,518. Thus, a powder
form of the tela submucosa can be implanted into a damaged or diseased
bone region for repair. The tela submucosa powder can be used alone, or
in combination with one or more additional bioactive agents such as
physiologically compatible minerals, growth factors, antibiotics,
chemotherapeutic agents, antigen, antibodies, enzymes and hormones.
Preferably, the powder-form implant will be compressed into a
predetermined, three-dimensional shape, which will be implanted into the
bone region and will substantially retain its shape during replacement of
the graft with endogenous tissues.

[0080]Tela submucosa of the invention can also be used as a cell growth
substrate, illustratively in sheet, paste or gel form in combination with
nutrients which support the growth of the subject cells, e.g. eukaryotic
cells such as endothelial, fibroblastic, fetal skin, osteosarcoma, and
adenocarcinoma cells (see, e.g. International Publication No. WO 96/24661
dated 15 Aug. 1996, publishing International Application No.
PCT/US96/01842 filed 9 Feb. 1996. In preferred forms, the tela submucosa
substrate composition will support the proliferation and/or
differentiation of mammalian cells, including human cells.

[0081]The inventive tela submucosa can also serve as a collagenous matrix
in compositions for producing transformed cells, (see, e.g.,
International Publication No. WO 96/25179 dated 22 Aug. 1996, publishing
International Application No. PCT/US96/02136 filed 16 Feb. 1996; and
International Publication No. WO 95/22611 dated 24 Aug. 1995, publishing
International Application No. PCT/US95/02251 filed 21 Feb. 1995). Such
compositions for cell transformation will generally include purified tela
submucosa of the present invention, for example in fluidized or paste
form, in combination with a recombinant vector (e.g. a plasmid)
containing a nucleic acid sequence with which in vitro or in vivo target
cells are to be genetically transformed. The cells targeted for
transformation can include, for example, bone progenitor cells.

[0082]Tela submucosa of the invention can also be used in body wall
repair, including for example in the repair of abdominal wall defects
such as hernias, using techniques analogous to those described in Ann.
Plast. Surg., 1995, 35:3740380; and J. Surg. Res., 1996, 60:107-114. In
such applications, preferred tela submucosa tissue grafts of the
invention promote favorable organization, vascularity and consistency in
the remodeled tissue. In dermatological applications, tela submucosa of
the invention can be used in the repair of partial or full thickness
wounds and in dermal augmentation using general grafting techniques which
are known to the art and literature (see, e.g. Annals of Plastic Surgery
1995, 35:381-388). In addition, in the area of burn treatment, it is
generally known to provide a dermal substitute onto which cultured
epidermal grafts (preferably cultured epidermal autografts, or CEA's) are
transplanted. Such cultured grafts have typically involved transplanting
keratinocytes and/or fibroblasts onto the dermal substitute. In
accordance with the present invention, the purified tela submucosa can be
used as the dermal substitute, for example in sheet form, and the CEA
accordingly transplanted onto the tela submucosa. In one mode of
practicing this aspect of the invention, keratinocytes can be
transplanted, for example by seeding or transferring a keratinocyte
sheet, onto the mucosal side of the tela submucosa. Fibroblasts can be
transplanted also on the mucosal and/or on the opposite (abluminal) side
of the tela submucosa.

[0083]Tela submucosa of the invention can also be used in tissue grafting
in urogenital applications. For instance, the tela submucosa can be used
in urinary bladder repair to provide a scaffold for bladder regeneration,
using techniques corresponding to those generally described in U.S. Pat.
No. 5,645,860; Urology, 1995, 46:396-400; and J. Urology, 1996, 155:2098.
In fluidized form, the inventive tela submucosa can also find use in an
endoscopic injection procedure to correct vesicureteral reflux. In such
applications, a submucosal injection can be made, for instance in the
area under the uretheral orifice of a patient, to induce smooth muscle
growth and collagen formation at the injection site.

[0084]In other areas, tissue graft constructs formed with tela submucosa
of the present invention can be used in neurologic applications, for
example in techniques requiring a dural substitute to repair defects due
to trauma, tumor resection, or decompressive procedures.

[0085]In order to promote a further understanding of the present invention
and its features and advantages, the following specific Examples are
provided. It will be understood that these specific Examples are
illustrative, and not limiting, of the present invention.

EXAMPLE 1

[0086]Thirty feet of whole intestine from a mature adult hog is rinsed
with water. This material is then treated in a 0.2 percent by volume
peracetic acid in a 5 percent by volume aqueous ethanol solution for a
period of two hours with agitation. The tela submucosa layer is then
delaminated in a disinfected casing machine from the whole intestine. The
delaminated tela submucosa is rinsed four (4) times with sterile water
and tested for impurities or contaminants such as endotoxins, microbial
organisms, and pyrogens. The resultant tissue was found to have
essentially zero bioburden level. The tela submucosa layer separated
easily and consistently from the whole intestine and was found to have
minimal tissue debris on its surface.

EXAMPLE 2

[0087]A ten foot section of porcine whole intestine is washed with water.
After rinsing, this section of tela submucosa intestinal collagen source
material is treated for about two and a half hours in 0.2 percent
peracetic acid by volume in a 5 percent by volume aqueous ethanol
solution with agitation. Following the treatment with peracetic acid, the
tela submucosa layer is delaminated from the whole intestine. The
resultant tela submucosa is then rinsed four (4) times with sterile
water. The bioburden was found to be essentially zero.

EXAMPLE 3

[0088]A small section of the tela submucosa intestinal collagen material
was subcutaneously implanted in a rat. Within 72 hours, significant
angiogenesis was observed.

EXAMPLE 4

[0089]Two sections of small intestine are processed by differing methods.
The first section is rinsed in tap water, disinfected for 2 hours in a
0.5% by volume aqueous ethanol solution comprising 0.2% by volume
peracetic acid, pH approximately 2.6, delaminated to the tela submucosa,
rinsed in purified water, divided into two samples and rapidly frozen.
The second section is rinsed in tap water, delaminated to the tela
submucosa, rinsed in purified water, placed in a 10% neomycin sulfate
solution for 20 minutes (as described in U.S. Pat. No. 4,902,508), rinsed
in purified water, divided into two samples and rapidly frozen. The four
above-prepared samples are tested for bioburden and endotoxin levels. The
first two samples each have bioburdens of less than 0.1 CFU/g and
endotoxin levels of less than 0.1 EU/g. The second two samples have
respective bioburdens of 1.7 CFU/g and 2.7 CFU/g and respective endotoxin
levels of 23.9 EU/g and 15.7 EU/g.

EXAMPLE 5

[0090]Three sections of small intestine are processed by differing
methods. The first is rinsed in tap water, disinfected for 2 hours in a
5% by volume aqueous ethanol solution comprising 0.2% by volume peracetic
acid, pH about 2.6, delaminated to the tela submucosa, rinsed in purified
water, and rapidly frozen. The second is rinsed in tap water, delaminated
to a tela submucosa, rinsed in purified water, disinfected according to
the methods of Example 1 in U.S. Pat. No. 5,460,962 (treatment for 40
hours in a 0.1% by volume aqueous solution of peracetic acid, buffered to
pH 7.2), and rapidly frozen. The third is rinsed in tap water,
delaminated to the tela submucosa, rinsed in purified water, disinfected
according to the methods of Example 2 in U.S. Pat. No. 5,460,962
(treatment in 0.1% by volume peracetic acid in high salt solution,
buffered to pH 7.2), and rapidly frozen. All three samples were tested
for endotoxins. The endotoxin levels were <0.14 EU/g for the first
sample, >24 EU/g for the second sample, and >28 EU/g for the third
sample.

EXAMPLE 6

[0091]Two sections of porcine small intestine were infected with
7×106 plaque forming units (PFU) of virus. Both were exposed
to a 0.18% peracetic acid, 4.8% aqueous ethanol solution at a nine-to-one
weight ratio of solution to material. A first sample was immersed in this
solution for 5 minutes; the second was immersed for 2 hours. The material
processed for minutes exhibited 400 PFU per gram of material. The
material processed for 2 hours exhibited zero PFU per gram of material.

EXAMPLE 7

[0092]Purified tela submucosa, prepared as described herein, was tested to
determine its nucleic acid content. Four samples of material weighing 5
mg each were subjected to DNA/RNA extraction as detailed in the DNA/RNA
Isolation Kit by Amersham Lifescience Inc., Arlington Heights, Ill.
Nucleic acid quantitation was performed by spectrophotometric
determination of solution optical densities at 260 nm and 280 nm. The
average nucleic acid content was 1.9±0.2 μg per milligram of
material.

[0093]Small intestinal submucosa, prepared as described by U.S. Pat. No.
4,902,508, was tested to determine its nucleic acid content. Four samples
of material weighing 5 mg each were subjected to DNA/RNA extraction as
detailed in the DNA/RNA Isolation Kit by Amersham. Nucleic acid
quantitation was performed by spectrophotometric determination of
solution optical densities at 260 nm and 280 nm. The average nucleic acid
content was 2.4±0.2 μg per milligram of material.

EXAMPLE 8

[0094]Sections of tela submucosa prepared according to the methods
described herein were sent to an independent testing laboratory (NamSA,
Inc., Northwood, Ohio) for biocompatibility testing as described in the
standard ISO 10993. The samples were tested for USP Acute Systemic
Toxicity, USP Intracutaneous Toxicity, Cytotoxicity, LAL Endotoxin,
material-mediated Pyrogenicity, Direct Contact Hemolysis, and Primary
Skin Irritation. The samples passed all tests, indicating that the
material is biocompatible.

[0095]It will be appreciated that variations of the above-described
processing procedures are intended to be within the scope of this
invention. For example, the source tissue for the tela submucosa, e.g.,
stomach, whole intestine, cow uterus and the like, can be partially
delaminated, treated with a disinfecting or sterilizing agent followed by
complete delamination of the tela submucosa. Illustratively, attached
mesentery layers, and/or serosa layers of whole intestine can be
advantageously removed prior to treatment with the disinfecting agent,
followed by delamination of remaining attached tissues from the tela
submucosa. These steps may or may not be followed by additional
disinfection steps, e.g., enzymatic purification and/or nucleic acid
removal. Alternatively, the tela submucosa source can be minimally
treated with a disinfecting or other such agent, the tela submucosa
delaminated from the tunica muscularis and tunica mucosa, followed by a
complete disinfection treatment to attain the desired contaminant
level(s). All such variations and modifications are contemplated to be a
part of the process described herein and to be within the scope of the
invention.

[0096]In addition, it will be appreciated that the publications cited
herein are indicative of the skill possessed by those practiced in the
relevant field, and each such publication is hereby incorporated by
reference in its entirety as if individually incorporated by reference
and fully set forth.